Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.
Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p1 (p1).Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7.
The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing RPS17 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.

File Name: Supplementary Movie 7:
Description: Nuclear localization of eYFP construct lysine mutant.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing lysine mutants of the RPS17 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.
Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the cons.TRIM22 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the h.TRIM22 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the dup1 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the dup2 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.
The protein structure of partial ORF1 including Kernow-C1-p1 HVR with artificial flexible linker, as well as Kernow-C1-p6 (RPS17 insertion), was predicted via AlphaFold2.Generated PDB files were analysed in PyMol.Depicted are the surface models of the PCP domain (red), the HVR (green) and the X-domain with helicase (blue).The 3D structure of the X-Domain with helicase were conserved and used to superimpose all structures.

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Name: Supplementary Movie 8: Description: Nuclear localization of eYFP SERPINA1.1 construct.Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the SERPINA1.1 insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.Huh7 cells were transfected with plasmids encoding a triple eYFP in tandem with the HVR of the construct Kernow-C1-p6 (p6) containing the SERPINA1.2insertion.Cells were fixed after 16-20 hours and the nucleus as well as the cell membrane were stained via immunofluorescence.The cells were imaged in 3D using a Zeiss Elyra 7. The cell and nuclear surface were reconstructed using Imaris 10.0.1 and the mean fluorescence intensity (MFI) for eYFP was measured for each compartment.Shown are example cells in 3D.eYFP is shown in green, the cell surface is depicted in red while the nuclear surface is depicted in light blue.